Promising antibacterial efficacy of arenicin peptides against the emerging opportunistic pathogen Mycobacterium abscessus

Background Mycobacterium abscessus, a fast-growing non-tuberculous mycobacterium, is an emerging opportunistic pathogen responsible for chronic bronchopulmonary infections in people with respiratory diseases such as cystic fibrosis (CF). Due to its intrinsic polyresistance to a wide range of antibiotics, most treatments for M. abscessus pulmonary infections are poorly effective. In this context, antimicrobial peptides (AMPs) active against bacterial strains and less prompt to cause resistance, represent a good alternative to conventional antibiotics. Herein, we evaluated the effect of three arenicin isoforms, possessing two or four Cysteines involved in one (Ar-1, Ar-2) or two disulfide bonds (Ar-3), on the in vitro growth of M. abscessus. Methods The respective disulfide-free AMPs, were built by replacing the Cysteines with alpha-amino-n-butyric acid (Abu) residue. We evaluated the efficiency of the eight arenicin derivatives through their antimicrobial activity against M. abscessus strains, their cytotoxicity towards human cell lines, and their hemolytic activity on human erythrocytes. The mechanism of action of the Ar-1 peptide was further investigated through membrane permeabilization assay, electron microscopy, lipid insertion assay via surface pressure measurement, and the induction of resistance assay. Results Our results demonstrated that Ar-1 was the safest peptide with no toxicity towards human cells and no hemolytic activity, and the most active against M. abscessus growth. Ar-1 acts by insertion into mycobacterial lipids, resulting in a rapid membranolytic effect that kills M. abscessus without induction of resistance. Conclusion Overall, the present study emphasized Ar-1 as a potential new alternative to conventional antibiotics in the treatment of CF-associated bacterial infection related to M. abscessus.


Background
Mycobacterium abscessus is a non-tuberculous mycobacterium whose incidence is increasing worldwide, and which in some industrialized countries even exceeds that of M. tuberculosis, the etiological agent of tuberculosis [1].Mycobacterium abscessus is responsible for severe lung infections and is associated with acute respiratory failure, notably in immunocompromised patients and/or patients with respiratory disorders such as cystic fibrosis (CF) [2,3].It is also responsible for cutaneous, bone and disseminated infections, and, in rare cases, for causing damage to the central nervous system [1].Furthermore, the presence of M. abscessus may evolve into severe complications after lung transplantation, limiting the therapeutic arsenal offered to CF patients [4].Mycobacterium abscessus exists as two phenotypically different morphotypes: a smooth (S) variant with a uniform, round and glossy appearance and that possesses glycopeptidolipids (GPL) at its surface; and an irregular and dry variant, called rough (R) variant, with no GPL at its surface thus resulting in cord formation [5,6].The S variant is associated with biofilm-like structures formation and should be responsible for the primo infection, whereas the R variant is usually found in severe and persistent forms of the clinical disease and represent the most virulent form of the bacteria [2,7].In addition, the naturally polyresistance of M. abscessus, not only to conventional anti-tuberculous drugs but also to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and β-lactams [1], leads to a treatment failure rate of nearly 60% [8].Whether intrinsic, adaptive or acquired, M. abscessus resistance to many antibiotics involves several mechanisms: low permeability of the cell wall, induction of antibiotic efflux pumps, mutations inactivating antibiotic-activating enzymes or activation of enzymes that neutralize antibiotics or modify their targets [9,10].Consequently, in order to avoid bacterial resistance, the treatment of M. abscessus infections implements a multitherapy possibly combined with surgery, resulting in an expensive and heavy treatment for the patient [11].New drugs as well as new strategies are thus urgently needed to fight infections due to M. abscessus, ideally with a different mechanism of action than conventional antibiotics in order to minimize and prevent resistance.
In this context, some antimicrobial peptides (AMPs) have demonstrated particularly interesting antibacterial activities against M. abscessus [12][13][14][15][16][17][18][19].AMPs are smallsized molecules (≤ 100 amino acids), with a cationic and a hydrophobic part [20].They play function in the symbiostasis and in the microbial clearance of a large variety of organisms, from unicellular (bacteria, archaea) as anti-competitors, to pluricellular (plants, fungi, invertebrates and vertebrates) as key components of the innate immune response [20,21].Often constituting the first line of defense, they possess a killing activity against a wide range of microorganisms from Gram-negative and Gram-positive bacteria, to fungi, enveloped viruses as well as parasites [22][23][24].More precisely, through their cationic part, AMPs interact with the anionic components at the surface of microorganisms, facilitating the insertion of the hydrophobic domain into the phospholipid bilayer thus inducing membrane destabilization and permeabilization resulting in bacterial lysis [20,21].AMPs can also target key intracellular processes that lead to pathogenic microorganism death without necessarily disrupting the membrane.Such processes include the inhibition of cell wall components expression, DNA or protein synthesis, protein folding, and metabolic turnover [25].Besides having a direct antimicrobial activity, several AMPs also possess the ability to modulate the host innate immune response and thereby indirectly to promote intracellular pathogen clearance [26].Given such diverse modes of action, few pathogens have developed resistance to AMPs which are also found active against pathogens already resistant to conventional antibiotics [27][28][29][30].
Arenicins exhibit antifungal and antibacterial activities against Gram-negative and Gram-positive bacteria including drug-resistant clinical isolates [33][34][35].They induce cell death in fungi as well as in bacteria via membrane permeabilization and disruption by formation of toroidal pores [33].It has been recently demonstrated that Ar-1 is also able to induce an apoptosis-like response in Escherichia coli-treated cells through accumulation of reactive oxygen species, imbalance of intracellular calcium concentrations, disruption of membrane potential, caspase-like activation, and DNA damage [37].Another important issue for the therapeutic potential of AMPs is related to their anti-inflammatory activity.Their potential role in inflammation may indeed result from the regulation of various processes such as cytotoxicity towards eukaryotic cells, complement activation, steroid synthesis, mast cell activation, monocyte chemo-attraction, and enhancement of cytokine expression [38].In the case of Ar-1, this peptide has been found to modulate complement activity in vitro depending on its concentration: stimulation at low concentrations (3.5-14.5 µM) and acting as a complement inhibitor at higher concentrations (29-58 µM) [39].
Finally, in contrast to many other antimicrobial peptides, such as β-defensins [40,41] and polymyxin B [32], arenicins are very stable AMPs that retain their antibacterial activity at high salt concentrations [42] and under high temperature conditions.A property probably due to the presence of one or two disulfide bonds along with several hydrogen bonds, allowing the formation of a ring that contributes to the conformational stability of the molecule (Fig. 1) [33,43].Considering that salt-induced inactivation of host defense peptides is an important issue in CF patients [44,45], the rapid and efficient killing of pathogenic bacteria in the presence of NaCl concentrations makes arenicin peptides particularly interesting lead structures for drug development.
From such physicochemical properties, together with their promising antibacterial activities against four pathogens commonly isolated from CF patients, namely Pseudomonas.aeruginosa, Staphylococcus aureus, Bacillus subtilis, and the yeast C. albicans [33]; we hypothesized that these three arenicin isoforms might be active against M. abscessus, the most drug-resistant mycobacterial species.
Hence, in this study we first compared the antimycobacterial activity, cytotoxicity and hemolytic activity of Ar-1, Ar-2 and Ar-3, as well as their respective disulfidefree derivatives (Fig. 1), with the aim of understanding the role of disulfide bridges in peptide function.To achieve this goal, the selective substitution of Cysteine residues by alpha-amino-n-butyric acid (Abu) was performed.Although the effects of replacing half-cystine residues in peptides and proteins with non-bridging residues such as alanine, valine, serine or threonine have been extensively studied, Abu has proved to be a more attractive substitutable residue because it is isosteric to half-cystine and has a similar polarity [46].As a result, such a Cys to Abu replacement has become the classic strategy used for decades to prevent the formation of disulfide linkage while leaving the peptide sequence globally unaltered [47][48][49].We further evaluated the potential of Ar-1 to permeabilize bacterial cytoplasmic membranes upon interaction with M. abscessus cells, and also investigated its binding affinity with monolayers of total lipids from M. abscessus.

Minimal inhibitory concentration (MIC) determination via the resazurin microtiter assay
For determining the antimicrobial activity of the different peptides, the Middlebrook 7H9 broth microdilution method was used in sterile 96-well flat-bottom Greiner Bio-One polypropylene microplates with lid (Thermo Fisher Scientific) using the resazurin microtiter assay (REMA) [50][51][52].Briefly, Mycobacterium abscessus was cultured up to mid log-phase (OD 600 ~ 1.5-2) and diluted to a cell density of 5 × 10 6 cells/mL in 7H9-S OADC .Then, 100 µL of this above inoculum was distributed to each well containing serial two-fold dilutions of arenicin, arenicin derivative or controls to a final volume of 200 µL.Growth controls containing no peptide (i.e., bacteria only = B), inhibition controls containing 50 µg/mL kanamycin (Euromedex, Souffelweyersheim, France) and sterility controls (i.e., medium only) without bacteria were included.After 5 days incubation at 37° C in a humidity chamber to prevent evaporation, 20 µL of a 0.025% (w/v) resazurin solution was added to each well.The microplate was re-incubated until appearance of a color change from blue to pink in the control well (i.e., bacteria without antibiotics).Fluorescence of the metabolite resorufin (λ ex / λ em = 530/590 nm) was measured using a Tecan Spark 10 M ™ multimode microplate reader (Tecan Group Ltd, France).Relative fluorescence units (RFU) were defined as: RFU% = (test well FU/mean FU of control B wells) × 100.MIC 50 and MIC 90 values were determined by fitting the RFU% sigmoidal dose-response curves in Kaleidagraph 4.2 software (Synergy Software, Reading, PA).The concentration of peptide leading to 50% and 90% inhibition of M. abscessus growth was defined as the MIC 50 and MIC 90 , respectively.All experiments were performed independently at least three times.

Cytotoxic assay on human cells
The toxicity of each peptide was evaluated on human cell lines using a resazurin assay as previously described [53,54].Human lung epithelial cells BEAS-2B (ATCC CRL-9609), liver cells HepG2 (ATCC HB-8065), and kidney cells A498 (ATCC HTB-44) were used.All cells were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS, Thermo Fisher Scientific), and 1% L-glutamine (Thermo Fisher Scientific) (DMEM FBS ) at 37 °C in a 5% CO 2 incubator to subconfluent concentrations.Cells were detached with trypsin-EDTA solution (Thermo Fisher Scientific), and around 1 × 10 4 cells/well were seeded in 96-well flat-bottom microplates (Greiner Bio-One) with a lid, and cultured for additional 48-72 h at 37 °C and 5% CO 2 until cell confluence.The medium was removed, and 100 µL of serial two-fold dilutions of the peptides in DMEM FBS were added to each well.Following a 48 h-incubation period at 37 °C and 5% CO 2 , cell viability was evaluated using the resazurin assay as described above.Fluorescence of the resazurin metabolite resorufin (λ ex /λ em = 530/590 nm) was measured after 2 h incubation at 37 °C and 5% CO 2 in the dark, with a microplate reader (Biotek, Synergy Mx, Colmar, France).The cytotoxic concentration of peptide leading to 50% cell death as compared to the control was defined as the CC 50 , and was determined by fitting the RFU% sigmoidal doseresponse curves in Kaleidagraph 4.2 software (Synergy Software).All experiments were conducted in triplicate.

Hemolytic activity assay
The hemolytic activity of each peptide was determined as previously described [55].Briefly, human erythrocytes (red blood cells, hRBC; Divbioscience, Ulvenhout, Netherlands) were washed 2-times with sterile phosphate buffered saline (PBS; Thermo Fisher Scientific) and pelleted by centrifugation at 800 rpm for 5 min; then resuspended in DMEM FBS to a final erythrocyte concentration of 8% (v/v).Then, 100 µL of this hRBC solution was distributed to each well of sterile 96-well flat-bottom microplates (Greiner Bio-One) containing serial two-fold dilutions of arenicin, arenicin-Abu derivative or controls in DMEM FBS to a final volume of 200 µL.
After 1 h incubation at 37 °C, microplates were centrifuged at 800 rpm for 5 min.Then, 100 µL of supernatant were carefully transferred to a new 96-well microplate, and OD 405 corresponding to the release of hemoglobin was measured using a microplate reader (Biotek, Synergy Mx, Colmar, France).DMEM FBS and Triton-X100 at 0.1% (v/v) were used as negative (i.e., 0% hemolysis) and positive (i.e., 100% hemolysis) controls, respectively.The concentration of peptide leading to 10% and 50% of hemolysis compared to the positive control was defined as the HC 10 and HC 50 , respectively, and was determined by fitting the dose-response curves in Kaleidagraph 4.2 software (Synergy Software).All experiments were done in triplicate.

Membrane permeabilization assay
For evaluating whether Ar-1 induces M. abscessus membrane permeabilization, bacterial cells in exponential growth phase were resuspended at 10 9 cells/ mL in 7H9-S OADC , before addition of 1 mg/mL propidium iodide (Sigma Aldrich, Lyon, France), a DNA/ RNA probe that is cell-impermeable.Then, 100 µL of this mix were distributed in the wells of a 96-well flatbottom Greiner Bio-One polypropylene microplate already containing Ar-1 at 4 × MIC 90 , 150 µM cetyltrimethylammonium bromide (CTAB) as positive control, or 7H9-S OADC for the negative control [56].Fluorescence (λ ex /λ em = 530/590 nm) was recorded at 5, 15, 30, 60 and 120 min with a Tecan Spark 10 M ™ multimode microplate reader (Tecan Group Ltd, France).Results were expressed in percentage of permeabilization compared to the maximum fluorescence obtained with CTAB, after removing the fluorescence of the negative control.All experiments were done independently at least three times.

Lipid insertion assay through surface pressure measurement
The insertion of Ar-1 into total lipids from M. abscessus was measured using reconstituted lipid monolayer at the air-water interface as previously described [56].Briefly, total lipids from M. abscessus S or R dry extracts were incubated at room temperature and under shaking with CHCl 3 -MeOH (1:2; v/v) for 16 h.Re-extraction of the residual lipids was done first with CHCl 3 -MeOH (1:1; v/v), and then with CHCl 3 -MeOH (2:1; v/v) during 16 h.The organic phases were pooled, concentrated under reduced pressure, re-suspended in CHCl 3 -MeOH (3:1; v/v), and then washed with 0.3% (w/v) NaCl solution.Centrifugation at 5000 rpm for 15 min allowed separation of the organic and aqueous phases.Then, the organic phase was dried over MgSO 4 .Extracted total lipids were dried, solubilized in CHCl 3 -MeOH (2:1, v/v), and stored at − 20 °C under nitrogen.
Surface pressure measurement was performed in a controlled atmosphere at 20 ± 1 °C using a fully automated microtensiometer (µTROUGH SX, Kibron Inc., Helsinki, Finland).This apparatus allowed the recording of the adsorption kinetics of a ligand onto a monomolecular film using a set of specially designed Teflon troughs.A few microliters of each total lipid extracts were spread using a 50 µL Hamilton's syringe at the surface of a sterile ultra-pure water (volume 800 µL) until the desired initial surface pressure (Π i ) was reached, thus generating a stable lipid monolayer at the air-water interface.The waiting time for the spreading solvent evaporation and for the lipid monolayer to reach equilibrium varied from 5 to 10 min depending on the spreading volume and the initial surface pressure.Ar-1 was further injected into the ultrapure water sub-phase below the stable monolayer, with a 10 µL Hamilton's syringe.The surface pressure increase (ΔΠ max ) due to the insertion of the peptide into the monolayer of total lipids was then continuously recorded until the equilibrium surface pressure was reached.The data were analyzed using the Filmware 2.5 program (Kibron Inc., Helsinki, Finland).

Electron microscopy
Cultures of M. abscessus S (10 9 cells/mL) were incubated with Ar-1 at 4 × MIC 90 for 120 min, then fixed with a solution of 2% glutaraldehyde in 200 mM HEPES (Thermo Fisher Scientific) (pH 7) directly in the culture medium (v/v) for 20 min at room temperature.After centrifugation and removal of the supernatant, the pellet was immediately covered with a second solution of 1% glutaraldehyde for 1 h at 4 °C.After four rinses of 5 min each with the HEPES buffer, the samples were post-fixed with 1% OsO 4 (Electron Microscopy Sciences, Hatfield, PA, USA) in H 2 O for 1 h at 4 °C.Then, four rinses of 5 min each were done with water.At this stage, the samples were gradually dehydrated using acetone-water mixtures of 50:50, 70:30, 90:10, and 96:4 (v/v), respecting a 5 min pause between each bath.Subsequently, the samples were transferred to 100% acetone and placed on a circular coverslip to complete the dehydration process.The coverslip was glued onto a nail-shaped support.Following carbonization, the samples were observed using the scanning electron microscope Tescan VEGA3 SBH (30 kV; Tescan, Brno-Kohoutovice, Czech Republic) and Jeol JSM-7900F (5 kV, 6 mm, LED detector) for higher magnification (Jeol, Croissy-sur-Seine, France).

Induction of resistance assay
Induction of bacterial resistance by Ar-1 was achieved by exposure of M. abscessus S and R to Ar-1 or to the control antibiotic clarithromycin (CLR) for 42 consecutive days, and with repeated broth microdilution susceptibility testing and subsequent MIC determination.After each MIC determination, the well containing the highest concentration of Ar-1 or CLR that allows bacterial growth was diluted 1:1000 in 7H9-S OADC and used as inoculum for the next MIC assay with fresh amounts of Ar-1 or CLR.

Statistical analysis
Graphpad Prism 8 (GraphPad Inc.) was used to perform all statistical analyses, which details are given in each figure legend.Differences were considered significant when the calculated p-values were smaller than 0.05.

Antimicrobial activity and cytotoxicity
Drug susceptibility testing of the arenicin and arenicin-Abu derivatives was assessed against both S and R variants of M. abscessus, with amikacin (AMK) as standard drug.The corresponding MIC 50 /MIC 90 values for each peptide, as determined by the REMA assay [51], are reported in Table 1.The three arenicin peptides displayed comparable antibacterial activity against M. abscessus S growth, with MIC 50 /MIC 90 values in the range 5.3-19.8µM and 12.2-29.3µM, respectively (Table 1 and Fig. 2).Considering the MIC values reached on M. abscessus R, they are 2.5-to eightfold greater for Ar-2 and Ar-3, respectively, than those obtained on the S variant (Table 1).In contrast, only Ar-1 was found to exhibit similar highly potent MIC 50/90 values against the two morphotypes (mean MIC 50 = 11.5 ± 1.0 µM/mean MIC 90 = 18.8 ± 0.9 µM).Furthermore, among the three native arenicin peptides tested, the fact that Ar-1 also displayed MIC 90 values comparable to those of the reference drug amikacin (AMK), made it the best growth peptide inhibitor of the two variants S and R of M. abscessus.
Replacement of all cysteines by Abu residues in the Ar-1-Abu, Ar-2-Abu and Ar-3-Abu resulted in a total loss of their antibacterial activity against the two M. abscessus variants (Table 1 and Fig. 2).Interestingly, the selective replacement of only two cysteines by Abu residues in Ar-3, likely to remove only one disulfide bond out of two, led to two different antibacterial behaviors.While Ar-3(7-16) exhibited MIC 50/90 values in the same order  and Ar-3, respectively) against M. abscessus S, with no real change in its antibacterial activity against the R variant compared to Ar-3 (Table 1 and Fig. 2).These results highlight the importance of the disulfide bonds (one for Ar-1/Ar-2 and two for Ar-3) which in such small peptides enables the integrity of the 3D structure to be maintained, and suggest that their presence is crucial for the antimycobacterial activity of each peptide against M. abscessus.
In parallel, the cytotoxic activity of the different peptides towards nontumorigenic human lung epithelial cells (BEAS-2B cells), human cells from a hepatocellular carcinoma (HepG2), and human kidney cells from a kidney cancer patient (A498) was investigated using a classical dose-response assay [57].The calculated response parameter was CC 50 , which corresponds to the concentration required to elicit a 50% decrease in cell viability in vitro, compared to the control.As seen in Table 2 and Fig. 3A, B, no toxicity towards BEAS-2B and HepG2 cells was observed, as demonstrated by CC 50 values > 100 µM.Regarding kidney A498 cells, although Ar-1 and Ar-1-Abu, and to a lesser extend Ar-2-Abu (CC 50 = 92.1 µM), were nontoxic, all other peptides displayed high toxicity with CC 50 values ranging from 36.6 to 46.7 µM (Table 2 and Fig. 3C).Such toxicity towards A498 cells was best characterized by calculating the respective therapeutic index [58] (TI A498 ); i.e., the ratio between CC 50 on A498 cells and MIC 50 on M. abscessus strains, for each peptide (Table 2).Accordingly, and except for Ar-1 (TI A498 > 8.6 for both variants) and Ar-3 (TIA 498 = 8.8 on M. abscessus S), all tested arenicin and arenicin-Abu peptides displayed TI A498 values < 2.2 synonymous with a very narrow window between efficacy and toxicity.
To better characterize the innocuity of the eight peptides, their hemolytic activity on human erythrocytes was further assessed through determination of their respective HC 50 and HC 10 values and calculation of their corresponding therapeutic indexes, TI H50 and TI H10 (Table 3 and Fig. 2D).First, in our experimental conditions, only Ar-2 was found to exert a strong hemolytic activity against red blood cells with a HC 50 of 29.2 µM resulting in TI H50 of 1.5 and 0.5 with regards to M. abscessus S and R variant, respectively (Table 3 and Fig. 2D).Considering their HC 10 values, except Ar-1-Abu and Ar-3(3, 20) totally inactive, all other arenicin and arenicin-Abu peptides tested showed variable hemolytic activities on human erythrocytes, resulting in predominantly low (< 0.4) and up to moderate (1-2.5)therapeutic indexes; with Ar-1 displaying the most promising TI H10 values (Table 3).
Overall, all these results clearly emphasized the efficacy of Ar-1 against M. abscessus S and R. Indeed, Ar-1 exhibited the most potent MIC values against both variants of M. abscessus (mean MIC 90 ~ 19 µM-Table 1), was nontoxic to host cells (CC 50 > 100 µM-Table 2), and showed only very weak hemolytic activity thus exhibiting the best therapeutic indexes related to hemolysis of human erythrocytes (TI H50 > ~ 8.7/TI H10 ~ 1.1-Table 3).Given all these findings, amongst the 8 tested peptides, Ar-1 was therefore found to display the best combination of antibacterial and cytotoxic properties and was then selected for further investigations.

Mechanism of action
The fact that Ar-1 is known to induce bacterial death through a pore forming mechanism in Gram-negative and -positive bacteria [33,42,43,[59][60][61][62][63] prompted us to evaluate its effect on membrane integrity of M. abscessus S and R using a propidium iodide assay (Fig. 4).Indeed, propidium iodide is a cell permeable dye which can enter bacteria only if the membrane is damaged, thus forming a highly fluorescent complex with DNA and RNA.As a consequence, the monitoring of propidium iodide fluorescence allows evaluation of membrane integrity.The membranolytic detergent cetyltrimethylammonium bromide (CTAB) was used as a positive control, corresponding to 100% permeabilization [15].
With the two S & R variants of M. abscessus, Ar-1 induced comparable permeabilization pattern over time (Fig. 4).As shown in Fig. 4, a plateau value corresponding to a non-significantly different membrane

Table 2 Evaluation of the cytotoxicity of the arenicin and arenicin-Abu peptides
All values are expressed as mean ± SD (n = 3) a CC 50 : Cytotoxic concentration of peptide leading to 50% cell toxicity in vitro, compared to the control b Therapeutic Index (TI) calculated by dividing the CC 50

Ar-1 interaction with monolayers of total lipids from M. abscessus
Given the ability of Ar-1 to cause membrane permeabilization in M. abscessus, its ability to insert into monolayers of total lipids from this bacterium [56] was further assessed (Fig. 5).In a first set of experiments, the dosedependent insertion of Ar-1 into total lipid monolayers was investigated (Fig. 5A).Total lipids were first spread until an initial surface pressure Π i = 30 ± 0.5 mN/m, corresponding to a lipid packing density theoretically equivalent to that of the outer leaflet of a cell membrane [64].
Increasing concentrations of Ar-1 were then injected into the aqueous phase below the lipid monolayer, and the variation in surface pressure due to the insertion of the peptide into the total lipid monolayer was continuously recorded until the new equilibrium surface pressure (ΔΠ max ) was reached (around 90 min-Fig.5A).
In agreement with propidium iodide assay results (Fig. 4), data showed that Ar-1 was able to insert into total lipid monolayers from both M. abscessus S and R with a maximal insertion reached at a concentration of 1.25 µM (i.e., ~ 0.11 × MIC 50 ), above which a plateau value in terms of ΔΠ max was obtained (Fig. 5A).Moreover, Ar-1 insertion resulted in a significantly higher equilibrium surface pressure, in the 1.25-10.0µM concentration range, with total lipid monolayers from M. abscessus S (ΔΠ max = 9.3 ± 0.69 mN/m) vs. M. abscessus R (ΔΠ max = 7.5 ± 0.65 mN/m; p-value < 0.001) (Fig. 5A).
In a second series of experiments, the influence of Π i [65] on the insertion process of Ar-1 into total lipid monolayers from M. abscessus was studied.The optimal final Fig. 4 Effect of Ar-1 on M. abscessus membrane integrity.M. abscessus S (blue circles) or R (green squares) variant was exposed to Ar-1 at 4 × MIC 90 concentration.The effect of Ar-1 on the integrity of the mycobacterial membrane was followed over 120 min using propidium iodide and compared to the maximum fluorescence obtained with 150 µM CTAB (100% permeabilization).Results are expressed as mean ± SD of three independent experiments.Statistical analysis was done using a non-parametric Mann-Whitney test using Prism 8.0 (Graphpad Inc.): ns, not significant (p > 0.05) Fig. 5 Insertion of Ar-1 into monolayers of total lipids from M. abscessus.A Variations of the surface pressure increase (ΔΠ max ) as a function of the peptide concentration upon Ar-1 insertion into total lipid monolayers from M. abscessus S and R. The increase in surface pressure due to the insertion of Ar-1 into the monolayers of total lipids, spread at an initial surface pressure Π i = 30 mN/m, was continuously monitored.Data are mean values ± SD of three independent experiments.Statistical analysis was done using a non-parametric Mann-Whitney test with Prism 8.0 (Graphpad Inc): ***p < 0.001.B Binding parameters of Ar-1 into a monomolecular film of total lipids from M. abscessus S and R. Variation of surface pressure (ΔΠ max ) after Ar-1 injection (1.25 µM final concentration) is represented as a function of the initial surface pressure, Π i = 10-30 mN/m peptide concentration of 1.25 µM, determined above by performing ΔΠ measurements at various Ar-1 concentrations, was used in these experiments.The plot ΔΠ max = f(Π i ) depicted in Fig. 5B, was first used to evaluate the binding parameters of Ar-1.Linear extrapolation to no increase in the surface pressure (ΔΠ max = 0) allowed to determine the critical surface pressure (Π c ), also named "maximum insertion pressure" [66], as being equal to 47.2 and 43.7 mN/m for M. abscessus S and R, respectively.Above this Π c value, which is specifically related to the peptide and the lipid forming the monolayer, no increase in the surface pressure will then occur.In addition, the synergy factor (slope of the linear regression + 1) which has been demonstrated to modulate protein/peptide insertion, as well as ΔΠ 0 (y-intercept of the curve corresponding to Π i = 0) [65][66][67] can also be deduced from the same plot.Indeed, and as defined by Salesse group [66,67], the observed positive synergy values (0.42-0.43) and the occurrence of a ΔΠ 0 value (25.3-26.9mN/m) lower than the related Π c (43-47 mN/m) are consistent with an 'insertion' surface pressure and favorable interactions between the peptide and the monolayer of total lipids from M. abscessus.

Microscopic observations
Mycobacterium abscessus S cultures were treated with Ar-1 at 4 × MIC 90 for 120 min, then fixed and processed for scanning electron microscopy experiments.As can be seen in Fig. 6, different modifications in the cell shape could be observed on Ar-1-treated M. abscessus S cells (Fig. 6B, D, F, and H) as compared to untreated one (Fig. 6A, C, E, and G).Indeed, bacteria incubated with Ar-1, especially when they are in clusters, seem to be covered by a sticky substance (Fig. 6B, D vs. A, C).Furthermore, clear alteration of cell wall morphology can be seen on single isolated bacteria treated with Ar-1 which seem to be empty with edges appearing blurred (see blue arrows in Fig. 6F and H).It should be noted that some cell fragments are also visible (see yellow arrowhead in Fig. 6H), thus strengthening the appearance of empty cells.In contrast, untreated control cells show a smooth and intact bacterial membrane (Fig. 6E and G).All these data clearly support disruption of the bacterial membrane through the pore forming activity of Ar-1 on M. abscessus S cells.

Induction of resistance assay
Regarding Ar-1, all results obtained above suggest that this peptide may target the bacterial membrane of M. abscessus.Given such membranolytic activity, Ar-1 should be less prompt at selecting resistant bacteria compared to conventional antibiotics targeting enzymes/ proteins for which mutations may induce resistance.This is especially true with M. abscessus, which is naturally resistant to many antibiotics and possesses the ability to acquire reversible and irreversible resistance to various antibiotics after a long period of treatment.In this context, we evaluated whether this mycobacterium could develop resistance to Ar-1 (Fig. 7).
As depicted in Fig. 7, a 42-days continuous exposure to Ar-1 induced no significant change in the MIC 90 value of this peptide against M. abscessus S and R variants, contrary to the conventional antibiotic clarithromycin (CLR) for which a 32-fold increase in MIC 90 values was observed after 20-30 days of exposure.

Discussion
Infections due to non-tuberculous mycobacteria represent a major public health problem worldwide, including developed countries where their incidence can even be higher than that of tuberculosis [68][69][70].Among nontuberculous mycobacteria, M. abscessus is a problem, causing skin infections as well as infections involving soft tissues [71,72], being increasingly prevalent during the last decade in CF patients [73,74].Yet, the treatment usually lasts 18 to 24 months with a cocktail of antibiotics, inducing severe adverse events in patients and also being a high cost for society [70,[75][76][77].Furthermore, the treatment outcomes are often disappointing with a cure rate of patients with a M. abscessus pulmonary infection of only 25-58% [76,78,79].This can be explained by several characteristics of M. abscessus, notably biofilm-like aggregation and intracellular survival, limiting antibiotic efficiency, as well as M. abscessus high resistance to many antibiotics [69,80].This resistance, whether intrinsic, adaptive or acquired, is such that M. abscessus is referred to as the "antibiotic nightmare" [10].As a consequence, new therapeutic tools, efficient against both M. abscessus S and R morphotypes, generating no resistance, are needed.
With no novel antibiotics entering the market in the past twenty years and the emergence of multidrug resistance (MDR) in many pathogenic bacteria, AMPs are making a comeback as the tool of choice against such pathogens.Indeed, thirty years after their discovery, a better understanding of their mode of action, their modification feasibilities and their synthesis at lower cost are reigniting the commercial development of AMPs [20,81].Among AMPs, arenicins are β-hairpin peptides stabilized by disulfide bonds which have been evidenced to exert antibiotic activities in vivo in mouse models of urinary tract infection, peritonitis and endotoxemia [82].Arenicin analogues are currently undergoing preclinical studies against infections caused by Gram-negative and Gram-positive MDR bacteria [35,83], thus highlighting Among these arenicin and arenicin-Abu peptides, we showed that Ar-1 displayed the best combination of antimycobacterial activity against both morphotypes (MIC 90 ~ 19 µM), cytotoxicity on eukaryotic cells and hemolytic activity towards human erythrocytes, resulting in relatively good therapeutic indexes [58] (Figs. 2, 3 and Table 1, 2).Concerning Ar-2 and Ar-3, they displayed good antibacterial activity against M. abscessus S-variant only, but also showed potent cytotoxicity against A498 cells and hemolytic activity (for Ar-2), which considerably limits their use in humans.Interestingly, the replacement of the two bridged Cys 3 & Cys 20 residues by Abu led to totally inactive and nontoxic peptides in the case of the Ar-1-Abu and Ar-2-Abu.Similar results were reported by Lee et al. [60] who found that a linear derivative of Ar-1, generated by substituting Cys 3 & Cys 20 with serine residues, was fourfold less active against Gram-negative bacterial cells than native Ar-1.In contrast, when the two cysteines were replaced by alanine residues, Krenev et al. showed that the resulting Ar-1(C/A) peptide was still able to form a twisted β-hairpin structure despite the absence of its disulfide bond, and also to retain both antibacterial and hemolytic activities as compared to natural peptide [84].From these biological data, it can be concluded that the hydrophobic-hydrophilic balance, the disulfide bridge, and the amphipathic β-sheet structure of the peptide play important roles in the biological activities of arenicins.Regarding Ar-3, neither the selective nor the complete replacement of the four cysteine residues improved the toxicity of the Ar-3(3-20), Ar-3(7-16) and Ar-3-Abu resulting peptides (Fig. 3 and Table 2).In contrast, the fact that Ar-3(7-16) retained similar antibacterial activity to Ar-3, whereas Ar-3-Abu was inactive and Ar-3(3-20) weakly active thus highlighted the importance of Cys 7 -Cys 16 bridge, which appears to drive the global antibacterial activity of all Ar-3 peptides probably by maintaining a correct folding of their respective 3D structure.
Such differences, however, between Ar-1 and Ar-2, were surprising as the two isoforms share 95% sequence identity and differ in only one residue: Val 10 in Ar-1 and Ile 10 in Ar-2 (Fig. 1).Such a single Val to Ile mutation has however been reported in the case of transthyretin transport protein, and has been shown to be responsible for a cardiomyopathy characterized by accelerated cardiac amyloid deposition [85].In a recent study on the link between arenicins' structure and antimicrobial and hemolytic activities [84], the authors suggested that the overall 3D conformation of Ar-1 could be described as more compact compared with the more elongated Ar-2 conformation.From our data, the specific position of the Val 10 and Ile 10 residues at the extremity of one β-sheet of Ar-1 and Ar-2, respectively, proved to be crucial for their inhibitory effect as well as for their cytotoxic and hemolytic activities.This is particularly true for the M. abscessus R variant, for which the MIC values of Ar-2 are ≥ 5 times higher than those of Ar-1 (Table 1).On the other hand, the effects of Ar-3 may be explained by its amino acid composition, which differs mainly from Ar-1 and Ar-2 with only 47% sequence identity.
Given their very limited application in humans due to lack of significant antibacterial activity combined with high toxicity to A498 cells, the use of Abu derivatives should be limited to comparative mechanistic studies with arenicin peptides.However, it might be interesting to test them on cancer cell lines other than A498 kidney cells to check whether these Abu-peptides have the same effect, which could pave the way for the possibility of a novel treatment for cancer cells.
We further focused on Ar-1 to better characterize its mechanism of action against M. abscessus S and R. Our data showed that this peptide provokes a ~ 42% mean permeabilization of M. abscessus membrane regardless of the S or R variant tested (Fig. 4).Moreover, Ar-1 was also able to efficiently interact and insert into monolayers of total lipids from M. abscessus S and R at high surface pressures: it was not excluded from the monolayer, and it exhibited a slightly higher affinity for lipid extracts from the S morphotype (Fig. 5).A recent study has underlined the importance of the surface-exposed glycopeptidolipids (GPL) present only in the S variant in M. abscessus hydrophobicity [86].By using quantitative imaging atomic force microscopy, the authors demonstrated that the transition from a smooth to a rough colony morphology, caused by the loss of GPL, led to a dramatic change in surface hydrophobicity, smooth bacteria displaying unusual nanodomains with varying degrees of hydrophobicity.In the case of Ar-1, the presence of GPL in M. abscessus S may alter cell-wall fluidity/permeability thus slightly favoring the insertion of this peptide at the origin of membrane permeabilization, cytoplasm leakage and bacterial cell lysis as depicted in Fig. 6.
As previously observed with another class of synthetic copolymers [56], such a pore-forming mode of action is likely to prevent the bacteria from developing any kind of resistance mechanism.This was clearly confirmed by the absence of appearance of Ar-1 resistance in M. abscessus S and R variants after a 42 days continuous exposure to this peptide (Fig. 7).
Overall, the fact that Ar-1 possesses good bactericidal activity against the S and R morphotypes of M. abscessus, without significant toxicity or hemolytic activity and without inducing resistance, is therefore very promising.Furthermore, since CF patients are initially infected with the S variant of M. abscessus, which can evolve into the R variant capable of forming cords and leading to severe and almost incurable lung infection, Ar-1 could represent a valuable alternative strategy to kill M. abscessus from the early stage of infection to the chronic lung infections.

Conclusion
In this article we have confirmed the great interest of Ar-1, a 21-residue antimicrobial peptide, in the fight against M. abscessus infections.Moreover, the fact that Ar-1 also exhibits a broad spectrum of activities against Gram-positive and Gram-negative bacterial as well as fungal pathogens [33], such as Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans, respectively, either alone or in combination with classical antibiotics as its synergistic activity has also been demonstrated [87], opens new perspectives for CF patients.Future studies should however be conducted, notably in vivo studies, to confirm the interest of this peptide in the therapeutic arsenal against bacterial infections in CF patients.In particular, to circumvent the described low hemolytic activity, encapsulation experiments might be done in order to use this peptide as an aerosol in human therapy, similar to amikacin liposome inhalation suspension [88], rather than injected into patients or administered orally.

45 Fig. 2
Fig. 2 Dose-response activity of the different arenicin and arenicin-Abu peptides against A M. abscessus S and B M. abscessus R strains replicating in broth medium, expressed as normalized relative fluorescence units (RFU%).Results are expressed as mean ± SD of at least three independent assays (n ≥ 3)

Fig. 3 Table 3
Fig. 3 Cytotoxic and hemolytic activity of the arenicin and arenicin-Abu peptides.A-C Cytotoxicity dose-response curves following 48 h-exposure to increasing concentrations of each peptide on human A BEAS-2B cells, B HepG2 cells, and C A498 cells.D Dose-response curves for human erythrocyte hemolysis after exposure to increasing concentrations of each peptide.Results are expressed as the mean ± SD of three independent experiments .0 ± 3.0% and 47.4 ± 16.0% (p > 0.05) was reached between 60-and 120-min incubation for M. abscessus S and R, respectively.Such a result is in line with the similar MIC 50/90 values obtained by Ar-1 against the two morphotypes.

Fig. 6
Fig. 6 Scanning electron microscopy of untreated control cells (left panels A, C, E, and G) and cells treated with Ar-1 at 4 × MIC 90 concentration for 120 min (right panels B, D, F, and H).Control cells appear well distinguishable, with clear cell contour.Cells treated with Ar-1, when in clusters, seem to be covered in a sticky substance (B and D).When isolated, cells treated with Ar-1, contrary to control cells, have blurred edges (blue arrow in F and H).Some cell pieces can also be seen in the treated sample (yellow arrowhead in H), contrary to the control one

Fig. 7
Fig. 7 Mycobacterium abscessus is not able to develop resistance mechanisms to Ar-1.The ability of M. abscessus A S or B R to develop resistance to Ar-1 was assessed by repeated MIC determination over 42 consecutive days exposure to this peptide.Clarithromycin (CLR) prompted to cause resistance was used as a positive control

Table 1
Antibacterial activities (µM) of arenicin and arenicin-Abu derivatives against M. abscessus S and R compared to standard drug Amikacin a value on A498 cell lines by the MIC 50 reached for each M. abscessus morphotype